Detection of Mycobacterium leprae DNA from Archaeological Skeletal Remains in Japan Using Whole Genome Amplification and Polymerase Chain Reaction

Koichi Suzuki,Kazuaki Nakamura,Kazunari Tanigawa,Mariko Sue,Akira Kawashima,Wataru Takigawa,Huhehasi Wu,Takeshi Akama,Shuichi Mori,Yuko Ishido,Norihisa Ishii,Aya Yoshihara,Immo A Hansen

doi:10.1371/journal.pone.0012422

Abstract

BackgroundIdentification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. However, it is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia.Methodology/Principal FindingsHere we demonstrate Mycobacterium leprae DNA from archaeological skeletal remains in Japan by polymerase chain reaction, DNA sequencing and single nucleotide polymorphism (SNP) analysis. In addition, we have established a highly sensitive method of detecting DNA using a combination of whole genome amplification and polymerase chain reaction, or WGA-PCR, which provides superior sensitivity and specificity in detecting DNA from trace amounts of skeletal materials.Conclusion/SignificanceWe have detected M. leprae DNA in archaeological skeletal remains for the first time in the Far East. Its SNP genotype corresponded to type 1; the first detected case worldwide of ancient M. leprae DNA. We also developed a highly sensitive method to detect ancient DNA by utilizing whole genome amplification.

Highlights

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) and has affected humans for millennia
We describe a highly sensitive whole genome amplification (WGA)-polymerase chain reaction (PCR) method that may be suitable for detecting trace amounts of ancient microbial DNA
We have demonstrated M. leprae DNA in excavated human skeletal remnants from Japan by both PCR analysis and DNA sequencing

Summary

Introduction

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) and has affected humans for millennia. Phylogeographic analysis of single nucleotide polymorphisms (SNPs) has revealed that M. leprae originated in Africa and spread to European and Asian countries and worldwide along with human migrations and trade routes. Such geographic and temporal migration has been demonstrated by SNPs analysis of ancient M. leprae DNA present in skeletal remains as old as 1,500 years ago [2,3]. Identification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. It is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia

Methods

Results

Conclusion